Kasap, C., Elemento, O., Kapoor, T.M. (2014), DrugTargetSeqR: a genomics- and CRISPR/Cas9-based method to analyze drug targets. Nature Chemical Biology. Summary: Determining a drug’s physiological target is a major challenge in chemical biology and drug discovery. Current strategies fall into two broad categories: model organism-based approaches and affinity-based methods (e.g. introducing a chemical handle for pull-downs followed by mass spectrometry). However, many drugs are not active in model organisms and affinity-based approaches are usually only effective when the drug is potent and the target is abundant in vivo. Further, establishing the physiological target of a drug depends on interpreting frequently unreliable correlations between protein knockdown phenotypes and chemical inhibition of activity. To address these limitations, we designed and validated a new approach at the interface of chemistry and genomics to identify a drug’s target in human cells. Briefly, transcriptomes are sequenced to identify genetic alterations (e.g. mutations) present in drug-resistant clones, but absent in the cell populations from which the clones are isolated. Genome editing is used to test if any of these genetic alterations is sufficient to confer drug resistance. Finally, biochemical analyses are used to establish the direct target. Our approach, which we named ‘DrugTargetSeqR’ (Seq: for sequencing, and R: CRISPR), has significant advantages over other target identification methods as the analysis is not biased to specific protein families (e.g. kinases), it does not require chemical modifications of the drug, and cell-type specific analysis is possible. Importantly, we can achieve ‘gold standard’ target validation, i.e. when a mutation in the target reduces drug sensitivity in both cellular contexts and biochemical assays. Application of this method also yields matched inhibitor-sensitive and resistant (via mutation in target) cell lines that can be employed to exclude any off-target effects when using chemical inhibitors to probe cellular mechanisms. Together, our findings indicate that DrugTargetSeqR is a powerful method that can uncover cellular mechanisms of resistance and reveal drug targets in human cells.